Nuclear localization sequences (NLSs) are relatively short sequences within a protein that direct its import into the nucleus. Specific NLS types are recognized with varying affinity by certain corresponding specific karyopherins, proteins which mediate the translocation of macromolecules across nuclear pore complexes. The yeast Saccharomyces has provided an excellent model system for the study of nuclear transport due to its advanced genetics, molecular biology, complete genome sequence (allowing the identification of any yeast protein by mass spectrometry) and the ease with which antigenic tags can be introduced into proteins to follow their localization and binding behavior. We have developed an "overlay assay" which depends on the fact that many NLSs can still be specifically recognized and bound by their karyopherins, even after the substrate protein has been denatured and partially renatured following SDS-PAGE and electrophoretic transfer to nitrocellulose. All impo rt substrates for karyopherins should by definition be at least present and usually enriched in nucleoplasm. Yeast is predicted to have a relatively low number of nuclear proteins (~1500) compared with "higher" eukaryotes. Thus yeast nucleoplasmic proteins were resolved by sequential cont.. hydroxylapatite-HPLC, RP-HPLC and SDS-PAGE such that the resulting Coomassie-stained gel bands each contained only one protein species, identifiable by mass spectrometry. A pilot scale project was initiated where separated material was immobilized on nitrocellulose and probed with functional tagged versions of two closely related karyopherins, Kap123p and Kap121p. Specifically recognized proteins were identified to generate a list of ~50 potential import substrates for these transport factors. Some 10 representative examples are being cloned as GFP chimeras and expressed in yeast containing mutant forms of karyopherins to confirm that their efficient nuclear localization is dependent upon these two karyopherins and thus that the overlay assay is a reliable test of karyopherin substrate specificity. Future directions will include the use of the assay, GFP constructs and tagged karyopherins to investigate the nature of this specificity.